{"paper_id":"064411d2-65d9-4abc-9ef4-c782878ea027","body_text":"Cannabidiol attenuates lipid metabolism and induces CB1 receptor-mediated ER stress associated apoptosis in ovarian cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cannabidiol attenuates lipid metabolism and induces CB1 receptor-mediated ER stress associated apoptosis in ovarian cancer cells Xuanhe Fu, Zhixiong Yu, Fang Fang, Weiping Zhou, Yuxin Bai, Zhongjia Jiang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5359456/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Ovarian cancer (OC) is the most deadly gynecological tumor. OC cells utilize cellular metabolic reprogramming to gain a survival advantage, particularly through aberrant lipid metabolic process. As the primary ingredient in exogenous cannabinoids, cannabidiol (CBD) has been shown to exert anticancer effects in several cancers. However, it is still unclear whether CBD can disrupt fatty acid metabolism and induce apoptosis in OC cells. In this study, we have demonstrated that CBD significantly inhibits the proliferation of OCs through a dependence on cannabinoid receptor type 1 (CB1R). Lipidomics and flow cytometry analysis revealed that CBD has the ability to decrease fatty acid levels and significantly suppress the transcription of genes involved in fatty acid uptake and synthesis in ES-2 cells. In addition, the analysis from RNA-seq and real-time RT-PCR revealed that CBD activated the endoplasmic reticulum (ER) stress pathway. Conversely, by supplementation with unsaturated fatty acid or blocking CB1R, ER stress or reactive oxygen species (ROS) signals with specific inhibitors could significantly relieve CBD induced a dose-dependent ER stress associated apoptosis, G0-G1 phase arrest, and mitochondrial dysfunction. Taken collectively, these data indicate that CBD may disrupt lipid metabolism, and lead to ER stress-related apoptosis in OCs. Our findings may provide a theoretical mechanism for anti-ovarian cancer using CBD. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Immunology Biological sciences/Molecular biology Health sciences/Molecular medicine Ovarian cancer Lipid metabolism Cannabidiol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction OC is the second most common malignant gynecological tumor worldwide, and it has the highest mortality rate and frequent relapses. Approximately 140,000 women globally die from OC each year [ 1 , 2 ]. Due to the lack of early-stage diagnostic methods, the 5-year survival rate is approximately 50% [ 3 ]. For treating OC patients, it is primarily based on surgical intervention followed by a combination of postoperative chemotherapy, immunotherapy, and radiotherapy [ 4 ]. However, these treatments have various limitations, including toxicity, chemoresistance and limited efficacy in OC. Thus, novel strategies are imperative to reduce the progression and mortality of OC. CBD is a non-psychoactive natural active compound derived from the medicinal plant cannabis ( Cannabis sativa L ). In 2018, the U.S. Food and Drug Administration approved it as a medication for the treatment of a rare form of epilepsy in children. CBD exerts broad pharmacological effects including neuroprotective, anti-epileptic, and anti-inflammatory effect [ 5 , 6 ]. Moreover, CBD has demonstrated a favorable safety profile and has exhibited antitumor effects in multiple types of cancer including ovarian, breast, colorectal, lung, and prostate cancers [ 7 , 8 ]. About 65 molecular targets for CBD have been reported with different targets responsible for different therapeutic effects of CBD [ 9 ]. In antitumor activity, CBD has the capacity to reduce the expression of the Id-1 gene through the regulation of ERK and ROS activation, consequently inhibiting the proliferation and invasion of breast cancer cells [ 10 ]. CBD is capable of inhibiting the growth and metastasis of breast cancer cells through the epidermal growth factor receptor pathway. In colorectal cancer cells, CBD triggers antitumor activity through a mechanism that is dependent on cannabinoid receptor 2 [ 11 ]. In addition, CBD can induce G0-G1 phase arrest and apoptosis of OC cells by upregulating LAIR-1 and blocking the PI3K/AKT/mTOR signaling pathways [ 12 ]. Recently, new strategies have emerged that target key enzymes involved in lipid uptake or synthesis within cancer cells. These approaches have demonstrated significant antitumor effects and are currently being developed for use in combination with chemotherapy or immunotherapy as potential interventions for cancer [ 13 ]. However, whether CBD can disturb fatty acid metabolism and induce apoptosis in OC cells remains unclear. Fatty acids play a crucial role as alternative energy sources for tumor cells, highlighting their significance in supporting the metabolic needs of cancer cells. The upregulation of fatty acid uptake and synthesis facilitates the proliferation and migration of tumor cells. At present, Limited information is available regarding the relationship between CBD-regulated lipid metabolism and apoptosis. Previous studies show that CBD increase ROS production and induce ER stress-associated apoptosis [ 11 , 15 ]. ER is a crucial cellular organelle that plays a significant role in regulating lipid metabolism and protein synthesis. ER stress can be induced by various physiological and pathological factors, such as the accumulation of misfolded or unfolded proteins, leading to the activation of the unfolded protein response (UPR) [ 16 ]. The UPR is initiated and regulated by three ER sensor proteins: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) [ 17 ]. Moderate activation of the UPR assists in addressing the accumulation of misfolded proteins and restoring ER homeostasis [ 18 ]. However, chronic and unresolved ER stress will ultimately result in cell apoptosis and cycle arrest through various pathways, such as c-Jun N-terminal kinase, C/EBP-homologous protein (CHOP), or caspase-12 [ 19 ]. Mitochondria play a pivotal role in energy metabolism and cellular physiological processes. Multiomics studies have revealed that mitochondrial dysfunction, oxidative stress, and apoptosis signaling pathways play critical roles in the survival and development of OC cells [ 20 ]. Any disturbance in the ER or mitochondria can mutually affect each other and trigger a cellular response. It has been reported that mitofusion 2 is an important link between ER stress and mitochondrial metabolism [ 21 ]. Additionally, CHOP activated by ER stress can also regulate mitochondrial dysfunction and the intrinsic apoptosis pathway involving Bcl2 family and caspase activation complex [ 22 ]. Numerous studies have increasingly demonstrated that dysregulation of lipid metabolism plays a significant role in tumor progression and the development of chemoresistance [ 23 ]. While, the ER is the primary site of lipid metabolism, it hosts numerous enzymes involved in lipid metabolism [ 24 ]. Stearoyl CoA desaturase 1 (SCD1) is an enzyme responsible for the key step in the conversion of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) within OC cells [ 25 ]. Meanwhile, SCD1 exhibited high levels of expression in ovarian cancer tissue and cell lines [ 13 ]. In a mouse model, treatment with SCD1 inhibitors has been shown to effectively inhibit the growth of OC stem cells [ 26 ]. Additionally, elevated levels of lipid unsaturation induced by SCD1 serve to protect cancer cells from ER stress and apoptosis [ 27 ]. These suggest SCD1 inhibition may prove to be effective approach for OC therapy. To better understand CBD-induced antitumor effect and the metabolic reprogramming mechanism in OC cells, we investigated the apoptosis signals and fatty acid content by transcriptomics and metabolomics after treatment with CBD. CBD could disorder lipid metabolism and initiate ER stress in OC cells. By supplementation with unsaturated fatty acid, oleic acid (OA) or blocking CB1R, ER stress or ROS signals with specific small-molecule inhibitor could significantly relieve CBD induced ER-mitochondrial associated apoptosis. Token together, these results suggested that CBD could disorder lipid metabolism, and lead to ER stress-associated apoptosis via CBR1-mediated way in OCs. Our findings may provide new clues into OC treatment for CBD. Materials and methods Reagents CBD was purchased from ZZStandards (Shanghai, China). Roswell park memorial institute (RMMI)-1640 and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Hyclone, Waltham, MA). The small-molecule inhibitors, including AM251, AM631, 4-PBA, and NAC, were purchased from Targetmol (MA, USA). OA and dimethyl sulphoxide (DMSO) were obtained from solarbio (Beijing, China). Cell cycle and apoptosis kit, as well as radio immunoprecipitation assay lysis buffer, were purchased from Beyotime (Jiangsu, China). The PE-conjugated CD36 and FITC annexin V apoptosis detection kit with propidium iodide (PI) were obtained from BioLegend (San Diego, CA, USA). BODIPY-labeled palmitate (BODIPY FLC16; Invitrogen) was utilized in conjunction with flow cytometry for the uptake experiment. The SYBR Premix Ex Taq Kit, Prime Script RT Master Kit, and the RNA-extracting reagent RNAiso Plus were procured from Takara Biotechnology (Dalian, China). Antibodies against CBR1, GRP78, ATF4, XBP1, CHOP, SCD1, Bcl2 and GAPDH were procured from ABclonal (Wuhan, China). Additionally, antibodies targeting mitofusion-2, cleaved Caspase 3, cleaved Caspase 8, cleaved Caspase 9, p65 and goat anti-rabbit IgG (conjugated to horseradish peroxidase) were obtained from Abcam. Cell line and culture condition Human ovarian SKOV3, ES-2, and Hey-A8 cells were obtained from the Cell Bank at the China Academy of Science (Shanghai, China) and cultured in RPMI 1640 with 10% FBS. The cells were incubated in a humidified incubator at 37°C with 5% CO2. Cell viability assay The cell viability assay was performed using the cell counting kit-8 (CCK-8 Kit) in accordance with the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). In brief, cells were seeded in 96-well plates at a density of 6 × 10 4 cells/well. After pre-treatment with small-molecule inhibitors or OA for 30 minutes, the cells were incubated with varying concentrations of CBD for 24 hours at 37°C and 5% CO2. DMSO was utilized as the negative control. The absorbance (Abs) was measured at 450 nm using a microplate reader. Tumor growth inhibition (%) was determined as follows: 1 - (Abs value in experimental groups / Abs value in negative control) × 100%. RNA-sequencing and DEGs analysis A total of 1 × 10 6 ovarian cancer cells were seeded in a 3.5 cm petridish in absence or presence of 40 µM CBD. The plates were incubated in a humidified atmosphere with 5% CO2 at 37°C for 24 h. Subsequently, the cells were collected and washed with cold phosphate buffered saline (PBS). All assays were conducted in triplicate. The samples underwent transcriptome sequencing treatment by Personal Biotechnology Co. (Shanghai, China). The edge R package ( http://www.rproject.org/ ) was employed to identify DEGs among various treatment groups [ 28 ]. The gene expression levels derived from RNA-Seq data were quantified using Transcripts Per Million reads (TPM), and then converted into Log2FC values, categorizing them into three groups. Genes with expression levels of Log 2 FC = 2, Log 2 FC < 2, and Log 2 FC > 2 were designated as showing no change, down-regulated change, and up-regulated change, respectively. A false discovery rate (FDR) value of ≤ 0.05 and |Log2FC| > 2 was employed to screen for significant DEGs. Gene ontology (GO) enrichment was conducted using DAVID (Database for Annotation, Visualization and Integrated Discovery). Quantitative real-time PCR (qRT-PCR) analysis After treatment with CBD and inhibitors as mentioned above, ES-2 cells were collected and total RNA was extracted from 8.4 × 10 5 cells using the RNA-extracting reagent RNAiso Plus. Subsequently, 0.5 µg of total RNA was reverse transcribed using a PrimeScript RT Master Kit following the manufacturer's instructions. The resulting cDNA was utilized for qRT-PCR analysis with an SYBR Premix Ex Taq Kit and ABI Prism 7000 (Applied Biosystems, Norwalk, CT). The PCR conditions were described in a previous study [ 29 ]. The sequences of all primers are listed in Table 1 [ 30 – 33 ]. Relative transcription levels were determined employing the 2 − ΔΔCt analysis method [ 34 ]. Table 1 Sequences for qRT-PCR primers Gene Forward Primer (5’ to 3’) Reverse Primer (3’ to 5’) Reference FABP5 5’-GGACAGCAAAGGCTTTGATG-3’ 5’-GCTCATTGAACTGAGCTTGG-3’ 30 CD36 5’-ATGTAACCCAGGACGCTGAG-3’ 5’-GTCGCAGTGACTTTCCCAAT-3’ 30 PPARγ 5’-AAGGCCATTTTCTCAAACGA-3’ 5’-GATGCAGGCTCCACTTTGAT-3’ 30 SREBP 5’-ACAGTGACTTCCCTGGCCTAT-3’ 5’-GCATGGACGGGTACATCTTCAA-3’ 31 FASN 5’-AAGGACCTGTCTAGGTTTGATGC-3’ 5’-TGGCTTCATAGGTGACTTCCA-3’ 31 SCD1 5’-TCTAGCTCCTATACCACCACCA-3’ 5’-TCGTCTCCAACTTATCTCCTCC-3’ 31 LDLR 5’-ACCAACGAATGCTTGGACAAC-3’ 5’-ACAGGCACTCGTAGCCGAT-3’ 31 ACACA 5’-ATGTCTGGCTTGCACCTAGTA-3’ 5’-CCCCAAAGCGAGTAACAAATTCT-3’ 31 GRP78 5’-CATCACGCCGTCCTATGTCG-3’ 5’-CGTCAAAGACCGTGTTCTCG-3’ 32 ATF4 5’-CGAGGTGTTGGTGGGGGACTTGA-3’ 5’-CAACCCATCCACAGCCAGCCATT-3’ 32 XBP1 5’-AACCTGTAGAAGATGACCTCGTTCC-3’ 5’-AAAGAGTTCATTGGCAAAAGTTCCAG-3’ 32 CHOP 5’-CCCTCACTCTCCAGATTCCAGTC-3’ 5’-CTAGCTGTGCCACTTTCCTTTCA-3’ 32 GAPDH 5’-TCAAGAAGGTGGTGAAGCAGG-3’ 5’-TCAAAGGTGGAGGAGTGGGT-3’ 32 ATF6 5’-CTGATGGCTGTTCAATACACAG-3’ 5’-GATCCCTTCGAAATGACACAAC-3’ 33 PERK 5’-CCAGTTTTGTACTCCAATTGCA-3’ 5’-CAGATACAGCTGGCCTCTATAC-3’ 33 Metabolic profiling The metabolic profiles of all samples were analyzed using the gas chromatograph/mass spectrometric (GC/MS) method, as described in previous studies [ 35 ]. The measurements were carried out by BioNovoGene Company (Jiangsu, China). Chromatographic and spectral analysis was performed using ChemStation and MassHunter (Agilent Technologies). Flow cytometry analysis Cell surface markers were determined by staining with fluorochrome-conjugated monoclonal antibodies (mAbs). The antibody panel included PE-conjugated CD36 for the analysis of lipoprotein receptors. Briefly, cells were incubated with inhibitors and CBD as indicated above. Subsequently, the cells (1 × 10 6 cells/tube) were resuspended in 50 µl of PBS containing the recommended concentrations of antibodies according to the manufacturer's instructions, and then incubated for 30 minutes at 4°C in the dark. Flow cytometry was conducted using a BD LSRFortessa, and the data was analyzed with FlowJo software. Evaluation of cell cycle distribution and cell apoptosis by flow cytometry Following treatment with CBD and the indicated inhibitors, PI staining was utilized for the analysis of DNA content. ES-2 cells were harvested and fixed with 70% ethanol at 4°C overnight. Subsequently, Cell cycle and apoptosis kit was applied to assess DNA content using flow cytometry (BD Biosciences LSRFortessa, USA). The distribution of cells in the G0, G1, S, G2, and M phases was analyzed using Modfit 5.0 software. The percentage of cells undergoing apoptosis was determined by double staining with the FITC annexin V apoptosis detection kit and PI. Treated ES-2 cells were collected and resuspended in annexin V binding buffer. A 100 µl cell suspension was transferred into a 1.5 ml tube, to which 5 µl of FITC annexin V and 10 µl of PI solution were added. After incubation for 15 minutes at 25°C in the dark, 300 µl of annexin V binding buffer was added to each tube. Flow cytometry was performed using a BD Biosciences LSRFortessa, and data were analyzed with FlowJo software. JC-1 mitochondrial membrane potential (MMP) assay The MMP was evaluated using a mitochondria staining kit (Boyotime, Shanghai, China). The cells were seeded on 12-well plates and incubated with 5 mM NAC in 96-well plates at 37°C and 5% CO2 for 30 min, followed by treatment with CBD at a final concentration of (30 µM, 50 µM) for 24 h. Subsequently, the cells were collected by centrifugation and resuspended in a staining solution containing 200 × JC-1 and 1 × staining buffer, then incubated at 37°C for 20 min. Finally, the cells were washed once with JC-1 buffer. Data analysis was performed using Flowjo software. The MMP depolarization was visualized using a Leika fluorescence microscope (Leika, Wetzlar, Germany). Determination of cellular ROS The intracellular levels of reactive oxygen species (ROS) were analyzed using a 2′,7′-dichlorofluorescin diacetate (H2DCFDA) cellular ROS detection assay kit (KeyGen Biotech Co., Nanjing, China). After treatment with NAC and CBD as indicated above, supernatants were removed from the treated cells and replaced with 5 µM DCFH-DA solution for 30 min at 37°C in the dark. After incubation, the cells were washed three times with PBS to remove residual particles, dead cells, and excess DCFH-DA probes. The fluorescence intensity of ROS was measured at Ex/Em 488/525 nm by a microplate reader. Western blotting After treatment with CBD and inhibitors as described above, a total of 4 × 106 ES-2 cells were collected. The cells were then lysed in RIPA lysis buffer at 4°C for 10 minutes to extract cellular protein. Samples containing equal amounts of protein (20–30 µg) were mixed with 5 × Laemmli buffer, boiled, and separated on 10–15% SDS-PAGE gels. Samples were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk, the primary antibody was incubated overnight at 4°C in an appropriate dilution. After washing, the blots were further incubated with HRP-conjugated secondary antibody for 1 h. Detection was performed using an enhanced chemiluminescence method. In our experiments, we used the following algorithm to evaluate the relative expression level of the target protein: Control group = [Control (Target protein/ Housekeeping protein) / Control (Target protein/Housekeeping protein)] = 1. Statistical analysis All values are presented as mean ± standard deviation (SD). The data were analyzed using a two-way analysis of variance (ANOVA) method. The post-hoc comparison was conducted using the follow-up least significant difference (LSD) test to assess differences between groups. Statistical significance was considered at a p value < 0.05 level. Results CBD inhibits OC cell proliferation via a CBR1-mediated mechanism First, we conducted the CCK-8 assay to investigate the impact of CBD on the viability of various types of OC cells (ES-2, SKOV3, Hey-A8). As illustrated in Fig. 1 A, CBD demonstrated significant antitumor activity in OC cells ( p < 0.05). Moreover, at the same concentration, the antitumor efficacy of CBD was notably higher in ES-2 cells compared to SKOV3 and Hey-A8 cells ( p < 0.05). The biological activity of CBD is known to be regulated by several receptors, including CBR1, CBR2, the transient receptor potential vanilloid subfamily 1 (TRPV1) receptor, and peroxisome proliferator-activated receptor γ (PPARγ) [ 36 ]. Next, we examined the relevance of these receptors to CBD-induced cell death by utilizing their selective inhibitors: AM251 (CB1 receptor antagonist), AM631 (CB2 receptor antagonist) [ 37 ], and Capsazepine (TRPV1 receptor antagonist) [ 38 ], and GW9662 (PPARγ inhibitor) [ 35 ]. We observed that the CBD-induced inhibition of cell viability was reversed by pre-treatment with AM251, but not by AM631. In addition, treatment with Capsazepine or GW9662 alone did not have any effect on the viability of the tested OC cells (data not shown). Additionally, we also examined the expression of CBR1 in OC cells. In the interim, we also conducted an investigation on the expression of CBR1 in OC cells. The protein level of CBR1 was found to be significantly higher in ES-2 cells compared to SKOV3 and Hey-A8 cells (Fig. 1 B and C). Additionally, we subjected ES-2 cells, which exhibited high sensitivity to CBD, to treatment with varying concentrations of CBD for 24 and 48 hours. The findings revealed that CBD (20–60 µM) significantly hindered the proliferation of ES-2 cells in a manner dependent on both time and dosage. Furthermore, an IC 50 value of 32 µM was observed after 24 hours of treatment (Fig. 1 D). These findings indicate that CBD may restrict the growth of OC cells in a manner dependent on CBR1. CBD induced ER stress in OC cells The DEGs were identified using RNA-seq. As illustrated in Fig. 2 A, a total of 2216 genes were found to be significantly upregulated, while 1908 genes were observed to be significantly downregulated following treatment with 40 µM CBD in ES-2 cells. Upon Go analysis, it was revealed that the upregulated differentially expressed genes (DEGs) were highly relevant to the ER-associated misfolded protein catabolic process and PERK-mediated unfolded protein response pathways (Fig. 2 B). We also observed that CBD-induced reduction in cell viability was reversed upon pre-treatment with 4-Phenylbutyric acid (4-PBA), a specific and irreversible inhibitor of ER stress, in a dose-dependent manner (Fig. 2 C). Subsequently, the transcriptome data was further validated using qRT-PCR, and the mRNA transcription levels of the genes related to ER stress were determined. The results demonstrated a significant increase in the mRNA transcription levels of glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), ATF6, PERK, ATF4, and CHOP in the treatment group compared to the control group ( p < 0.05). The qRT-PCR results for the CBD-induced ER stress-related genes were in accordance with the findings from RNA-Seq analysis (Fig. 2 D). To investigate the intrinsic mechanism of this effect, western blot analysis was conducted. The findings indicated that CBD could significantly increase the mRNA transcription and protein expression of ER stress-related markers in a dose-dependent manner ( p < 0.05). 4-PBA effectively inhibited the upregulation of ER stress-related markers induced by CBD in a dose-dependent manner (Fig. 2 E-I). These findings suggest that CBD may suppress the growth of OC cells through the ER stress pathway. CBD induces OC cells apoptosis and cell cycle arrest by regulating the ER stress signaling To further investigate the correlation between apoptosis and CBD-induced ER stress in OC cells, Annexin V/PI staining was assessed using flow cytometry assay. Figure 3 A and B illustrate that CBD can effectively induce apoptosis in ES-2 cells in a manner that is dependent on dosage ( p < 0.05). Furthermore, the addition of the ER stress inhibitor, 4-PBA groups, significantly prevented CBD-induced apoptosis. The observed increase in apoptosis due to CBD was consistent with an enhanced inhibition of tumor cell growth. We utilized flow cytometry to further assess the impact of CBD on the distribution of cell cycles in OC cells. The exposure of these cells to CBD led to a dose-dependent increase in the percentage of cells in the G0-G1 phase (Fig. 3 C and D). Furthermore, the additional 4-PBA groups significantly rescued the CBD-induced G0-G1 phase cycle arrest of OC cells ( p < 0.05). Based on these above-mentioned results, we have confirmed that CBD is capable of inducing apoptosis in OC cells and arresting the G0-G1 phase cycle through the activation of ER stress signaling. CBD causes lipid metabolism disorder and induces ER stress response Fatty acids serve as an important carbon source in the tumor microenvironment, and fatty acid-mediated lipid metabolism plays crucial roles in the survival of ovarian cancer cells [ 39 ]. Elevated levels of unsaturated fatty acids (UFAs) have been shown to provide protection for cancer cells against apoptosis induced by ER stress [ 27 ]. To investigate the impact of fatty acid metabolism on CBD-induced apoptosis in OC cells, we initially conducted a GC-MS analysis to assess the levels of lipid metabolites in CBD-treated OC cells. As illustrated in Fig. 4 A, the group treated with 40 µM CBD showed significantly higher levels of saturated fatty acids (SFAs), including myristic acid (14C:0), palmitic acid (16C:0), and stearic acid (18C:0) compared to the control group ( p < 0.05). Simultaneously, the levels of UFAs' content, particularly palmitoleic acid (C16:1), heptadecenoic acid (C17:1), and oleic acid (C18:1), were found to be significantly reduced following treatment with CBD in ES-2 cells ( p < 0.05). Furthermore, the analysis using Go revealed that the downregulated DEGs are highly associated with the UFA metabolic process and fatty acid biosynthetic process (Fig. 4 B). The results of qPCR analysis demonstrated that the CBD-treated group was able to reduce the transcription levels of genes associated with fatty acid uptake, including fatty acid binding protein 5 (FABP5), low-density lipoprotein receptor (LDLR), and PPARγ. The proportion of CD36-positive events was reduced following treatment with CBD (Fig. 4 C and D). Additionally, the transcription levels of genes related to fatty acid biosynthesis, such as acetyl-CoA carboxylase alpha (ACACA), Fatty acid synthase (FASN), SCD1, and sterol regulatory element binding protein 1 (SREBP), were also decreased post-CBD treatment (Fig. 4 D). Additionally, we investigated whether CBD could inhibit the uptake of fatty acids from the extracellular environment using fluorescently labeled palmitate (BODIPY FLC16) [ 40 ]. The findings indicated that the CBD treatment group significantly inhibited the uptake of palmitate (Fig. 4 E). Previous research has indicated that elevated levels of saturated fatty acids (SFAs), as a result of inhibiting stearoyl-CoA desaturase 1 (SCD1), can induce ER stress and apoptosis in several types of cancer. Furthermore, ER stress could be effectively alleviated through supplementation with UFAs [ 27 ]. In this study, we observed that the addition of exogenous oleic acid could effectively alleviate the CBD-induced antitumor activities in OC cells in a dose-dependent manner (Fig. 4 F). CBD significantly inhibited the gene transcription and protein expression of SCD1 in a dose-dependent manner. Additionally, treatment with CBD resulted in a significant relief of both mRNA transcription and protein expression of ER stress-related markers by oleic acid in a dose-dependent manner ( p < 0.05, Fig. 4 G-N). Altogether, these data suggested that CBD interfered with lipid metabolism leading to ER stress in OC cells. Oleic acid rescued CBD-directed ER stress-mediated apoptosis and cell cycle arrest To determine whether UFAs provide broad protection against CBD-induced ER stress-related apoptosis, We utilized Annexin V/PI staining to assess the levels of apoptosis in ES-2 cells. The results are presented in Fig. 5 A and B, CBD-treated group significantly increase apoptosis rates including both early and late apoptosis compared with the control group in serum-free medium. The CBD-induced apoptosis was significantly inhibited by supplying with OA ( p < 0.05). Furthermore, exogenous OA had a marked effect on restoring the cell cycle porfile of CBD-treated cells in a dose-dependent manner ( p < 0.05, Fig. 5 C and D). From these results above, our findings have confirmed that the imbalance between unsaturated and saturated fatty acids is the primary cause of CBD-induced ER stress-related apoptosis in OC cells. CBD induce lipid metabolism disorder and ER stress via a CBR1-mediated mechanism Previous research has demonstrated that several crucial pathways involved in the growth, differentiation, and metabolism of tumor cells interact with CBR signaling [ 41 , 42 ]. To examine whether CBD-induced fatty acid metabolism disorder and ER stress are CBR1-mediated, we used AM251, the antagonist of CBR1, to suppress the activity of CBR1 in ES-2 cells. As illustrated in Fig. 2 C, the CBD-induced inhibition of cell viability was found to be dose-dependently reversed by pre-treatment with AM251. Meanwhile, AM251 significantly rescued apoptosis and G0-G1 phase arrest of CBD-treated cells ( p < 0.05, Fig. 3 A-D). In qPCR analyses, the treatment with CBD resulted in a significant dose-dependent decrease in the transcription levels of genes associated with fatty acid metabolism ( p < 0.05). The downregulation of those genes were restored after blocking the CBR1 signaling with AM251 (Fig. 6 A). The balance of SFAs and UFAs is regulated by SCD1 to finely tune the functions of ER stress [ 27 ]. Subsequently, we investigated the levels of gene transcription and protein expression associated with the ER response after blocking the CBR1. The upregulation of mRNA transcription and the protein expression of ER stress-related markers were significantly alleviated by AM251 ( p < 0.05, Fig. 6 B-I). These findings suggest that CBD possesses the capability to regulate disruption of lipid metabolism in OC cells and reduce UFA synthesis through the CBR1-SCD1 signaling pathway, ultimately resulting in ER stress-induced apoptosis. CBD induced mitochondrial dysfunction and cascade-mediated apoptotic pathways Alteration of ER stress markers is a key indicator of mitochondrial stress [ 43 ]. Based on the above findings, we hypothesized that CBD may induce cytotoxicity by targeting the mitochondria. CCK-8 and annexin V staining results indicated that the inhibitory effects of CBD on cell viability and apoptosis were significantly attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), a dose-dependent scavenger of reactive oxygen species (ROS) ( p < 0.05, as shown in Fig. 2 C, 3 A and B). Previous research has demonstrated that levels of MMP and ROS are commonly utilized as markers to assess mitochondrial function [ 44 ]. To further investigate the effect of CBD on mitochondrial function, we first detected the MMP by the fluorescent probe JC-1. Comparative to the control group, treatment with CBD led to an increase in JC-1 monomer green fluorescence and a decrease in JC-1 aggregation (red fluorescence), resulting in a higher JC-1 monomer ratio. The flow cytometry results demonstrated that treatment of ES-2 cells with 30 and 50 µM CBD led to a significant increase in the loss of MMP by 8.4 and 14.5-fold respectively, compared to DMSO-treated control cells. Furthermore, intracellular levels of ROS in CBD-treated ES-2 cells increased by up to 1.3 and 1.8-fold, respectively, in a dose-dependent manner compared to control cells. Similarly, the decrease in MMP and the generation of ROS could be significantly reversed by NAC treatment (Fig. 7 A-C). In addition, western blot revealed a significant downregulation of mitofusion 2 and antiapoptotic Bcl2 in the high CBD group, while the expression of cleaved Caspase-8, Caspase-9, and Caspase-3 was significantly upregulated (Fig. 7 D-I). Taken together, our data showed that CBD may trigger mitochondrial dysfunction and apoptosis in OC cells. Discussion CBD is a non-addictive compound found in the medicinal plant Cannabis. Most previous studies have primarily focused on the protective effect of the nervous system, due to its ability to easily pass through the blood-brain barrier [ 45 ]. Additionally, current evidence has demonstrated that CBD can inhibit the proliferation and metastasis of tumor cells, as well as induce apoptosis and autophagy in various types of cancer including breast, colorectal cancer, and gastric cancer [ 7 , 11 ]. These studies also demonstrated that the involvement of ER stress or oxidative stress in the induction of cell death following treatment with CBD. CBD acts its effects mainly through the expression of CBD receptors on the cells. Significantly, high levels of CBR1 expression have been observed in ovarian cancer (OC) tissue, and the expression of CB1R is positively correlated with the level of malignancy in OCs [ 46 ]. Therefore, as a specific CBR1 agonist with efficacy against OC, CBD could be effectively utilized for OC therapy. In this study, we have observed high expression of CBR1 in all three types of human OC cells. However, a receptor-mediated mechanism contributing to the promising antitumor activity of CBD in OC cells has not yet been elucidated. The results of our study demonstrate a significant difference in the CBD-induced antitumor effect on OC cell lines ES-2, SKOV3, and Hey-A8. The inhibitory effects of CBD on ES-2 cells were found to be significantly higher (45.1%) compared to SKOV3 (25.46%) and Hey-A8 (21.29%). Additionally, the western blot results demonstrated a significantly higher protein level of CBR1 in ES-2 cells compared to that in SKOV3 and Hey-A8 cells. After inhibition of CBR1 by AM251, the CBD-induced repression of cell viability was significantly reversed. These results suggest that CBD may inhibit the growth of OC cells in a CBR1-mediated manner. To further elucidate the antitumor mechanism of CBD in OC cells, we initially utilized transcriptome analysis to examine the DEGs in ES-2 cells. Go analysis results suggest that CBD-induced antitumor effect was highly relevant to the ER-associated misfolded protein catabolic process. In addition, the gene transcript and expression levels for ER stress were significantly upregulated after treatment with CBD. Blocking ER stress response with 4-PBA, we revealed that CBD-induced OC cells apoptosis and G0-G1 phase cycle arrest relied on activation of XBP1 and ATF4/CHOP signaling pathways. Fatty acids play a crucial role as important energy sources and serve as a key component of phospholipids in cell membranes [ 14 ]. In OCs, the accumulation of UFAs has been shown to support cell growth, as well as increase cancer cell migration and invasion [ 47 ]. Lipid metabolism plays a complex role in multiple cell death pathways, including necroptosis and ferroptosis [ 48 ]. Recently, Zhao et al. revealed that controlling the balance between SFAs and UFAs can initiate ER stress in tumors [ 27 ]. These findings suggest that regulating lipid metabolism in OCs may be a potential therapeutic strategy. To test this prediction, using GC-MS and RNA-seq analysis, we observed that the CBD group significantly downregulated the UFA metabolic process and decreased the transcription levels of genes associated with fatty acid uptake and synthesis in OC cells (Fig. 3 ). CD36 regulates cellular energy homeostasis and intracellular cholesterol in OC cells [ 49 , 50 ]. Likewise, CD36 expression was significantly decreased after treatment with CBD. In addition, we found that CBD could convert SFAs to UFAs and palmitic and stearic acid to palmitoleic and OA, respectively. The regulation of the conversion from saturated fatty acids to unsaturated fatty acids is mediated by SCD1 [ 51 ]. Meanwhile, SCD1 and its predominant product, oleic acid, have been shown to have beneficial effects in restoring ER homeostasis, promoting cell cycle progression, and enhancing proliferation [ 52 ]. This conclusion is further supported by our results, exogenous oleic acid could significantly relieve CBD-induced antitumor activities, apoptosis and G0-G1 phase arrest. Based on the combined results of qPCR and western blot analysis, it was observed that CBD-induced mRNA transcription and protein expression of ER stress-related markers could be significantly mitigated in a dose-dependent manner by exogenous oleic acid. These results strongly imply that CBD may initiate ER stress-associated apoptosis by disrupting lipid metabolism. Over the past decade, numerous studies have demonstrated that CB1 and CB2 receptor agonists can function as direct antitumor agents by activating ERK, p38 MAPK, and JNK1/2 pathways [ 53 , 54 ]. However, a clear distinction was observed between the activity produced and the specific cancer cell line studied. Furthermore, numerous studies have suggested that CBD may hinder the viability of cancer cells through mechanisms that bypass the activation of cannabinoid receptors. Ramer et al. reported that CBD induced PPARγ-dependent toxicity in lung cancer cells [ 55 ]. Another study found CBD mediated endothelial cell autophagy and apoptosis via ROS-mediated heme oxygenase-1, but not by the CBD-activated receptors (CBR1, CBR2) [ 29 ]. A recent investigation found that TRPV2 is a target of cannabinoids and is involved in CBD-induced autophagic death of glioma stem-like cells [ 56 ]. In breast cancer cell lines, CBD induced ER stress through the TRPV1 receptor-dependent signaling pathway by increasing Ca 2+ influx [ 15 ]. These results all show that CBD can modulate certain pathways involved in cancer development and exert their antitumor effects. Our study revealed that CBD exhibited very effective antitumor activity in OC cells and significantly downregulates the transcription of genes related to fatty acid metabolism via CBR1 signaling. Notably, we also observed that CBD significantly decreased the protein expression of p65, which is necessary for regulating SCD1 activity [ 26 ]. The downregulation of CBD-induced p65 and SCD1were also relieved by blocking CBR1. Similarly, the upregulation of mRNA transcription and protein expression of ER stress-related markers was significantly alleviated by AM251. In accordance with our findings, a previous study has reported that increased expression levels of SCD1 protected ovarian cancer cells from apoptosis induced by ER stress [ 27 ]. Collectively, these results suggest that CBD may regulate lipid metabolism via CBR1/SCD1 signaling pathway to modulate ER stress-triggered apoptosis in OC cells. Mitochondria are essential for generating energy and play a critical role in maintaining cell survival and promoting metastatic evasion [ 57 ]. Mitochondrial dysfunction, which results in depolarized MMP and decreased ATP levels, suppresses OC progression [ 58 ]. Previous studies showed that mitochondria and ER crosstalk was also important in regulating cell metabolism and cell death [ 59 ]. The process of CHOP-mediated apoptosis involves the regulation of Bcl2 family proteins, leading to the induction of mitochondrial outer membrane permeabilization and caspase-dependent cell death [ 60 , 61 ]. In our study, flow cytometry analysis demonstrated that CBD could dose-dependently increase the loss of mitochondrial membrane potential (MMP). The decrease in MMP will consequently result in an elevation of the intracellular ROS level. As shown in Fig. 7 C, the level of ROS content in the CBD-treated group was found to be higher than that in the control group. Once the MMP is fully depolarized, it will trigger caspase-dependent apoptosis pathways [ 62 ]. This process occurs within the mitochondria and plays a crucial role in programmed cell death. The conclusions were consistent with our western blot results. In this study, the expression levels of Bcl2 and mitofusion 2 were significantly downregulated and the pro-apoptotic protein caspase-8, -9, and − 3 were significantly upregulated after treatment with CBD in ES-2 cells. Meanwhile, CBD-induced mitochondria dysfunction and the expression of apoptotic proteins could be significantly reversed by NAC. These results indicate that CBD may trigger OC cells apoptosis through the ER-mitochondrial pathway. Conclusions In summary, our results demonstrated that CBD promoted OC cells apoptosis and G0-G1 phase arrest by disrupting the CBR1-mediated lipid metabolism and ER stress- and mitochondrial dysfunction-associated apoptosis signaling pathways (Fig. 8 ). These findings could potentially contribute to the development of CBD as a pharmaceutical for treating OC. Declarations Conflicts of interest The authors declare no financial or commercial conflict of interest. Author Contribution Conceptualization, X.F., and G.L.; Formal analysis, X.F., Z.Y., Y.B., W.Z., B.Y., Y.S., Z.J., and X.T.; Funding acquisition, G.L.; Investigation, X.F., Z.Y., X.T., Z.J., and B.Y.; Methodology, X.F., G.L. and Z.Y.; Project administration, G.L.; Software, X.F., F.F., Z.Y., B.Y. Y.S. and X.T.; Supervision, G.L.; Validation, G.L.; Writing – original draft, X.F.; Writing – review & editing, G.L. 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Supplementary Files WBoriginaldata.pdf Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Dec, 2024 Reviews received at journal 17 Dec, 2024 Reviews received at journal 28 Nov, 2024 Reviews received at journal 28 Nov, 2024 Reviewers agreed at journal 27 Nov, 2024 Reviewers agreed at journal 27 Nov, 2024 Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 15 Nov, 2024 Editor invited by journal 13 Nov, 2024 Submission checks completed at journal 13 Nov, 2024 First submitted to journal 30 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5359456\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":383952434,\"identity\":\"f4acf028-d7a2-4ea7-a982-e3851b8b8153\",\"order_by\":0,\"name\":\"Xuanhe Fu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xuanhe\",\"middleName\":\"\",\"lastName\":\"Fu\",\"suffix\":\"\"},{\"id\":383952435,\"identity\":\"f43f2a0c-6492-4035-b4a1-acfedbfc5d7b\",\"order_by\":1,\"name\":\"Zhixiong Yu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhixiong\",\"middleName\":\"\",\"lastName\":\"Yu\",\"suffix\":\"\"},{\"id\":383952436,\"identity\":\"129bc0c1-9474-44ea-9ac0-13d57b6eacac\",\"order_by\":2,\"name\":\"Fang Fang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fang\",\"middleName\":\"\",\"lastName\":\"Fang\",\"suffix\":\"\"},{\"id\":383952437,\"identity\":\"1bb05bad-7b18-4906-8768-f5f0103e4494\",\"order_by\":3,\"name\":\"Weiping Zhou\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Weiping\",\"middleName\":\"\",\"lastName\":\"Zhou\",\"suffix\":\"\"},{\"id\":383952438,\"identity\":\"5e021343-d10d-4994-a210-8c711a5a5901\",\"order_by\":4,\"name\":\"Yuxin Bai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuxin\",\"middleName\":\"\",\"lastName\":\"Bai\",\"suffix\":\"\"},{\"id\":383952439,\"identity\":\"798014c0-6c57-445d-8a91-a9f72e84fd48\",\"order_by\":5,\"name\":\"Zhongjia Jiang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhongjia\",\"middleName\":\"\",\"lastName\":\"Jiang\",\"suffix\":\"\"},{\"id\":383952440,\"identity\":\"c444325d-f00a-4709-8bf2-da0759087288\",\"order_by\":6,\"name\":\"Biao Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Biao\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"},{\"id\":383952441,\"identity\":\"bb337d55-fd91-4706-bca2-81133029b59d\",\"order_by\":7,\"name\":\"Ye Sun\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ye\",\"middleName\":\"\",\"lastName\":\"Sun\",\"suffix\":\"\"},{\"id\":383952442,\"identity\":\"dca013cc-b24c-4997-9310-c23cc6301935\",\"order_by\":8,\"name\":\"Xing Tian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xing\",\"middleName\":\"\",\"lastName\":\"Tian\",\"suffix\":\"\"},{\"id\":383952443,\"identity\":\"4e324ab3-2ab2-4626-9e26-d0bb5c8ad4e6\",\"order_by\":9,\"name\":\"Guangyan Liu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACPmYEm/EBlGGAVwsbkhZmmFICWpDZEsRpYecxfs1Tc8duw/Gzxyp/tm2TZ2Bv3ibBUHMHj8N4zKx5jj1L3nAmL+02b9ttwwaeY2USDMee4dVizMN2ONngQI7Zbca22wkMEjlmEowNhwlo+QfUcv6NWeFPkBb5NwS1GD/mbTtsZ3Ajx4yBF2wLDyEtbGWMc/sOJ0jeeGMszXPutmEbT1qxRcIx3Fr4+Q9v/vDm22F7vvM5hh9/lN2W52c/vPHGhxrcWhig0ZG44ACQZGSDxlQCPg3ASP8AJOzlG0DsP/iVjoJRMApGwcgEAHVGUQ+TFP21AAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Shenyang Medical College\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Guangyan\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-10-30 08:08:06\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5359456/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5359456/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-025-88917-1\",\"type\":\"published\",\"date\":\"2025-02-05T15:56:59+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":70759253,\"identity\":\"cb512cd1-03e4-41a7-a71e-83e0ba5a7519\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:05:56\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1254691,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD repressed viability of OC cells by a CBR1-dependent mechanism. (A) OC cells were pre-incubated with the respective receptor antagonist AM251 or AM631 for 30 min at a final concentration of 100 μM and then further co-incubated with CBD (40 μM) for another 24 h. The cell inhibition rate was detected by CCK-8 assay. (B and C) Western blot analysis was conducted to detect CB1R expression in SKOV3, Hey-A8, and ES-2 cells. (D) The ES-2 cells were treated with gradient concentrations of CBD (10 μM-60 μM) for 24 and 48 h, respectively, and the cell viability was assessed by CCK-8 assay. DMSO was used as a negative control. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/9919f16f0dbd44ec312e7d81.png\"},{\"id\":70760399,\"identity\":\"513a7d3e-7618-4c4e-a873-de512038a2af\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:21:56\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2830071,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD could effectively induce ER stress in OC cells. (A) DEGs identified in ES-2 cells before and after being treated with 40 μM CBD. Volcano plot for the DEGs. Red, blue and gray points represent up-regulated, down-regulated, and non-regulated DEGs, respectively. (B) GO enrichment analysis of the upregulated DEGs before and after being treated with 40 μM CBD. The most significant GO terms were those with corrected \\u003cem\\u003ep\\u003c/em\\u003e-value of \\u0026lt; 0.05. The rich factor represents the number of DEGs that exist in this term accounting for the total number of genes of this term. (C) ES-2 cells were pre-incubated with 4-PBA, AM251 or NAC for 30 min at indicated concentrations. Cells were subsequently co-incubated with CBD at indicated concentrations. After 24 h, the cell inhibition rate was detected by CCK-8 assay. (D) ES-2 cells were pre-incubated with 4-PBA for 30 min at a final concentration of 500 μM and then further co-incubated with CBD at indicated concentrations. After 24 h, total cellular RNA was extracted and reverse transcribed. The mRNA transcription levels of ER stress-related genes GRP78, XBP1, ATF6, PERK, ATF4, and CHOP were detected by qRT-PCR as described in methods. Heatmap represents log2 values of relative mRNA transcription levels (see color scale). The log2 value of each gene in control cells was set to 0. Untreated cells served as a negative control. (E-I) The protein expression levels of the ER stress-related factors were detected by western blot analysis. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/718bb390abf410e4595e47af.png\"},{\"id\":70759252,\"identity\":\"793d480e-02ca-4f6c-bd2f-7c98a2f23b57\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:05:56\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4438188,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD induced apoptosis and G0-G1 cell cycle arrest by regulating the ER stress signaling. (A and B) Flow cytometric analysis of Annexin V-FITC/PI-stained cells. ES-2 cells were pre-incubated with 4-PBA, AM251 or NAC for 30 min at indicated concentrations. Cells were subsequently co-incubated with CBD at indicated concentrations. After 24 h, cells had been harvested and stained for analysis. Dot plots of total events are shown with frequencies of cells in each quadrant. (C and D) Cell cycle distribution was determined by flow cytometry. ES-2 cells were pre-incubated with 4-PBA or AM251 for 30 min at indicated concentrations. Cells were subsequently co-incubated with CBD at indicated concentrations. After 24 h, cells were harvested and fixed with 70% ethanol at 4 °C overnight, then, stained with PI. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/e5328cfb2e85d5b4a84106a5.png\"},{\"id\":70760122,\"identity\":\"7f140f0d-c5a0-4aa0-be18-42d923321832\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:13:56\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4736287,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD disrupted lipid metabolism and leads to ER stress in OC cells (A) Lipid metabolism was identified in ES-2 cells before and after being treated with 40 μM CBD. Log2 values for each metabolite represent the average of duplicates. (B) GO enrichment analysis of the downregulated DEGs. The most significant GO terms were those with corrected P-value of \\u0026lt; 0.05. The rich factor represents the number of DEGs that exist in this term accounting for the total number of genes of this term. (C) ES-2 cells were stained with PE-conjugated CD36 Abs before analysis by flow cytometry. Dot plots of total events are shown with frequencies of cells in each quadrant. (D) Total cellular RNA was extracted and reverse transcribed. The mRNA transcription levels of Lipid metabolism-related genes SCD1, FABP5, LDLR, FASN, ACACA, SREBP, and PPARγ were detected by qRT-PCR. (E) Representative plots of BODIPY FLC16 in ES-2 cells were collected and analyzed by flow cytometry after being treated with 40 μM CBD for 24 h. Untreated cells served as a negative control. (F) ES-2 cells were co-incubated with CBD at indicated concentrations for 24 h, in presence or absence of 25 μM OA. The cell inhibition rate was detected by CCK-8 assay. (G)The mRNA transcription levels of ER stress-related genes were detected by qRT-PCR.Heatmap represents log2 values of relative mRNA transcription levels (see color scale). The log2 value of each gene in control cells was set to 0. (H-N) The protein levels of GRP78, ATF4, XBP1, CHOP, SCD1, and p65 were detected by western blot analysis. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/0aba9f8ffb7c8b2f85c78e02.png\"},{\"id\":70759258,\"identity\":\"f6bd8cf3-d415-4042-ab83-8655acd56f33\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:05:56\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3543161,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eOA reversed CBD induced apoptosis and G0-G1 cell cycle arrest. (A and B) Flow cytometric analysis of Annexin V-FITC/PI-stained cells. ES-2 cells were co-incubated with CBD at indicated concentrations for 24 h, in presence or absence of 25 μM OA. Dot plots of total events are shown with frequencies of cells in each quadrant. (C and D) Cell cycle distribution was determined by flow cytometry. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/71c1fd17a22e7fdbd34f2685.png\"},{\"id\":70760124,\"identity\":\"57666f5f-6ebc-4506-94bf-2ace80cf8139\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:13:56\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4448479,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBR1 regulated lipid metabolism and ER stress in OC cells. (A and B) ES-2 cells were pre-incubated with AM251 for 30 min at a final concentration of 100 μM and then further co-incubated with CBD at indicated concentrations. After 24 h, the mRNA transcription levels of lipid metabolism- and ER stress-related genes were detected by qRT-PCR. Heatmap represents log2 values of relative mRNA transcription levels (see color scale). The log2 value of each gene in control cells was set to 0. Untreated cells served as a negative control. (C-I) The protein levels of GRP78, ATF4, XBP1, CHOP, SCD1, and p65 were detected by western blot analysis. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/b01f1287cb392ac145e88897.png\"},{\"id\":70760400,\"identity\":\"5182597c-bff9-4766-8ae5-b9b7400cf95b\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:21:56\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":7488987,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD triggers mitochondrial dysfunction and apoptosis in ES-2 cells. (A and B) Changes in MMP in ES-2 cells were determined based on JC-1 fluorescence. ES-2 cells were pre-incubated with NAC for 30 min at a final concentration of 5 mM and then further co-incubated with CBD at indicated concentrations for 24 h. The effect of CBD on the disruption of MMP was detected by JC-1 assay. Relative JC-1 red/green population is represented as a bar graph in percentage-ratio under the flow cytometry data. (C) The fluorescence intensity of ROS was measured by microplate reader. (D-I) Protein levels of mitofusion, cleaved caspase 3, 8, 9 and Bcl2 were detected by western blot analysis. Each value indicates the mean ± SD of results obtained from three independent experiments. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/0228ccc139b8b82e9d616ee4.png\"},{\"id\":70759256,\"identity\":\"f7a1f6dd-4a84-4b44-bdfc-6b7fa48b8f54\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:05:56\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":197513,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCBD inhibits lipid metabolism and induces ER stress- and mitochondrial dysfunction-associated apoptosis in OC cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/e37e1aa9d4125b7d28e48907.png\"},{\"id\":75929970,\"identity\":\"f70bb664-5362-430d-9339-2b7cd14e9c1b\",\"added_by\":\"auto\",\"created_at\":\"2025-02-10 16:08:17\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":40146971,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/e627a506-23c8-4667-928f-7e78d7ef2e93.pdf\"},{\"id\":70759260,\"identity\":\"6638de19-ba4e-4639-8388-9a522f76ceaf\",\"added_by\":\"auto\",\"created_at\":\"2024-12-06 11:05:56\",\"extension\":\"pdf\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":582214,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"WBoriginaldata.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5359456/v1/28c7074cd0bb91fdb7f9cdb2.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Cannabidiol attenuates lipid metabolism and induces CB1 receptor-mediated ER stress associated apoptosis in ovarian cancer cells\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eOC is the second most common malignant gynecological tumor worldwide, and it has the highest mortality rate and frequent relapses. Approximately 140,000 women globally die from OC each year [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Due to the lack of early-stage diagnostic methods, the 5-year survival rate is approximately 50% [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. For treating OC patients, it is primarily based on surgical intervention followed by a combination of postoperative chemotherapy, immunotherapy, and radiotherapy [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. However, these treatments have various limitations, including toxicity, chemoresistance and limited efficacy in OC. Thus, novel strategies are imperative to reduce the progression and mortality of OC.\\u003c/p\\u003e \\u003cp\\u003eCBD is a non-psychoactive natural active compound derived from the medicinal plant cannabis (\\u003cem\\u003eCannabis sativa L\\u003c/em\\u003e). In 2018, the U.S. Food and Drug Administration approved it as a medication for the treatment of a rare form of epilepsy in children. CBD exerts broad pharmacological effects including neuroprotective, anti-epileptic, and anti-inflammatory effect [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Moreover, CBD has demonstrated a favorable safety profile and has exhibited antitumor effects in multiple types of cancer including ovarian, breast, colorectal, lung, and prostate cancers [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. About 65 molecular targets for CBD have been reported with different targets responsible for different therapeutic effects of CBD [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. In antitumor activity, CBD has the capacity to reduce the expression of the Id-1 gene through the regulation of ERK and ROS activation, consequently inhibiting the proliferation and invasion of breast cancer cells [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. CBD is capable of inhibiting the growth and metastasis of breast cancer cells through the epidermal growth factor receptor pathway. In colorectal cancer cells, CBD triggers antitumor activity through a mechanism that is dependent on cannabinoid receptor 2 [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. In addition, CBD can induce G0-G1 phase arrest and apoptosis of OC cells by upregulating LAIR-1 and blocking the PI3K/AKT/mTOR signaling pathways [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Recently, new strategies have emerged that target key enzymes involved in lipid uptake or synthesis within cancer cells. These approaches have demonstrated significant antitumor effects and are currently being developed for use in combination with chemotherapy or immunotherapy as potential interventions for cancer [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. However, whether CBD can disturb fatty acid metabolism and induce apoptosis in OC cells remains unclear.\\u003c/p\\u003e \\u003cp\\u003eFatty acids play a crucial role as alternative energy sources for tumor cells, highlighting their significance in supporting the metabolic needs of cancer cells. The upregulation of fatty acid uptake and synthesis facilitates the proliferation and migration of tumor cells. At present, Limited information is available regarding the relationship between CBD-regulated lipid metabolism and apoptosis. Previous studies show that CBD increase ROS production and induce ER stress-associated apoptosis [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. ER is a crucial cellular organelle that plays a significant role in regulating lipid metabolism and protein synthesis. ER stress can be induced by various physiological and pathological factors, such as the accumulation of misfolded or unfolded proteins, leading to the activation of the unfolded protein response (UPR) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. The UPR is initiated and regulated by three ER sensor proteins: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Moderate activation of the UPR assists in addressing the accumulation of misfolded proteins and restoring ER homeostasis [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. However, chronic and unresolved ER stress will ultimately result in cell apoptosis and cycle arrest through various pathways, such as c-Jun N-terminal kinase, C/EBP-homologous protein (CHOP), or caspase-12 [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Mitochondria play a pivotal role in energy metabolism and cellular physiological processes. Multiomics studies have revealed that mitochondrial dysfunction, oxidative stress, and apoptosis signaling pathways play critical roles in the survival and development of OC cells [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Any disturbance in the ER or mitochondria can mutually affect each other and trigger a cellular response. It has been reported that mitofusion 2 is an important link between ER stress and mitochondrial metabolism [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Additionally, CHOP activated by ER stress can also regulate mitochondrial dysfunction and the intrinsic apoptosis pathway involving Bcl2 family and caspase activation complex [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eNumerous studies have increasingly demonstrated that dysregulation of lipid metabolism plays a significant role in tumor progression and the development of chemoresistance [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. While, the ER is the primary site of lipid metabolism, it hosts numerous enzymes involved in lipid metabolism [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Stearoyl CoA desaturase 1 (SCD1) is an enzyme responsible for the key step in the conversion of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) within OC cells [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Meanwhile, SCD1 exhibited high levels of expression in ovarian cancer tissue and cell lines [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. In a mouse model, treatment with SCD1 inhibitors has been shown to effectively inhibit the growth of OC stem cells [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Additionally, elevated levels of lipid unsaturation induced by SCD1 serve to protect cancer cells from ER stress and apoptosis [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. These suggest SCD1 inhibition may prove to be effective approach for OC therapy.\\u003c/p\\u003e \\u003cp\\u003eTo better understand CBD-induced antitumor effect and the metabolic reprogramming mechanism in OC cells, we investigated the apoptosis signals and fatty acid content by transcriptomics and metabolomics after treatment with CBD. CBD could disorder lipid metabolism and initiate ER stress in OC cells. By supplementation with unsaturated fatty acid, oleic acid (OA) or blocking CB1R, ER stress or ROS signals with specific small-molecule inhibitor could significantly relieve CBD induced ER-mitochondrial associated apoptosis. Token together, these results suggested that CBD could disorder lipid metabolism, and lead to ER stress-associated apoptosis via CBR1-mediated way in OCs. Our findings may provide new clues into OC treatment for CBD.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eReagents\\u003c/h2\\u003e \\u003cp\\u003eCBD was purchased from ZZStandards (Shanghai, China). Roswell park memorial institute (RMMI)-1640 and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Hyclone, Waltham, MA). The small-molecule inhibitors, including AM251, AM631, 4-PBA, and NAC, were purchased from Targetmol (MA, USA). OA and dimethyl sulphoxide (DMSO) were obtained from solarbio (Beijing, China). Cell cycle and apoptosis kit, as well as radio immunoprecipitation assay lysis buffer, were purchased from Beyotime (Jiangsu, China). The PE-conjugated CD36 and FITC annexin V apoptosis detection kit with propidium iodide (PI) were obtained from BioLegend (San Diego, CA, USA). BODIPY-labeled palmitate (BODIPY FLC16; Invitrogen) was utilized in conjunction with flow cytometry for the uptake experiment. The SYBR Premix Ex Taq Kit, Prime Script RT Master Kit, and the RNA-extracting reagent RNAiso Plus were procured from Takara Biotechnology (Dalian, China). Antibodies against CBR1, GRP78, ATF4, XBP1, CHOP, SCD1, Bcl2 and GAPDH were procured from ABclonal (Wuhan, China). Additionally, antibodies targeting mitofusion-2, cleaved Caspase 3, cleaved Caspase 8, cleaved Caspase 9, p65 and goat anti-rabbit IgG (conjugated to horseradish peroxidase) were obtained from Abcam.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eCell line and culture condition\\u003c/h3\\u003e\\n\\u003cp\\u003eHuman ovarian SKOV3, ES-2, and Hey-A8 cells were obtained from the Cell Bank at the China Academy of Science (Shanghai, China) and cultured in RPMI 1640 with 10% FBS. The cells were incubated in a humidified incubator at 37\\u0026deg;C with 5% CO2.\\u003c/p\\u003e\\n\\u003ch3\\u003eCell viability assay\\u003c/h3\\u003e\\n\\u003cp\\u003eThe cell viability assay was performed using the cell counting kit-8 (CCK-8 Kit) in accordance with the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). In brief, cells were seeded in 96-well plates at a density of 6 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well. After pre-treatment with small-molecule inhibitors or OA for 30 minutes, the cells were incubated with varying concentrations of CBD for 24 hours at 37\\u0026deg;C and 5% CO2. DMSO was utilized as the negative control. The absorbance (Abs) was measured at 450 nm using a microplate reader. Tumor growth inhibition (%) was determined as follows: 1 - (Abs value in experimental groups / Abs value in negative control) \\u0026times; 100%.\\u003c/p\\u003e\\n\\u003ch3\\u003eRNA-sequencing and DEGs analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eA total of 1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e ovarian cancer cells were seeded in a 3.5 cm petridish in absence or presence of 40 \\u0026micro;M CBD. The plates were incubated in a humidified atmosphere with 5% CO2 at 37\\u0026deg;C for 24 h. Subsequently, the cells were collected and washed with cold phosphate buffered saline (PBS). All assays were conducted in triplicate. The samples underwent transcriptome sequencing treatment by Personal Biotechnology Co. (Shanghai, China).\\u003c/p\\u003e \\u003cp\\u003eThe edge R package (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.rproject.org/\\u003c/span\\u003e\\u003cspan address=\\\"http://www.rproject.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) was employed to identify DEGs among various treatment groups [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. The gene expression levels derived from RNA-Seq data were quantified using Transcripts Per Million reads (TPM), and then converted into Log2FC values, categorizing them into three groups. Genes with expression levels of Log\\u003csub\\u003e2\\u003c/sub\\u003eFC\\u0026thinsp;=\\u0026thinsp;2, Log\\u003csub\\u003e2\\u003c/sub\\u003eFC\\u0026thinsp;\\u0026lt;\\u0026thinsp;2, and Log\\u003csub\\u003e2\\u003c/sub\\u003eFC\\u0026thinsp;\\u0026gt;\\u0026thinsp;2 were designated as showing no change, down-regulated change, and up-regulated change, respectively. A false discovery rate (FDR) value of \\u0026le;\\u0026thinsp;0.05 and |Log2FC| \\u0026gt; 2 was employed to screen for significant DEGs. Gene ontology (GO) enrichment was conducted using DAVID (Database for Annotation, Visualization and Integrated Discovery).\\u003c/p\\u003e\\n\\u003ch3\\u003eQuantitative real-time PCR (qRT-PCR) analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eAfter treatment with CBD and inhibitors as mentioned above, ES-2 cells were collected and total RNA was extracted from 8.4 \\u0026times; 10\\u003csup\\u003e5\\u003c/sup\\u003e cells using the RNA-extracting reagent RNAiso Plus. Subsequently, 0.5 \\u0026micro;g of total RNA was reverse transcribed using a PrimeScript RT Master Kit following the manufacturer's instructions. The resulting cDNA was utilized for qRT-PCR analysis with an SYBR Premix Ex Taq Kit and ABI Prism 7000 (Applied Biosystems, Norwalk, CT). The PCR conditions were described in a previous study [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. The sequences of all primers are listed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e [\\u003cspan additionalcitationids=\\\"CR31 CR32\\\" citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Relative transcription levels were determined employing the 2\\u0026thinsp;\\u0026minus;\\u0026thinsp;ΔΔCt analysis method [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSequences for qRT-PCR primers\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGene\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eForward Primer (5\\u0026rsquo; to 3\\u0026rsquo;)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eReverse Primer (3\\u0026rsquo; to 5\\u0026rsquo;)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eReference\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFABP5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GGACAGCAAAGGCTTTGATG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GCTCATTGAACTGAGCTTGG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCD36\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-ATGTAACCCAGGACGCTGAG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GTCGCAGTGACTTTCCCAAT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePPARγ\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-AAGGCCATTTTCTCAAACGA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GATGCAGGCTCCACTTTGAT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSREBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-ACAGTGACTTCCCTGGCCTAT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GCATGGACGGGTACATCTTCAA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFASN\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-AAGGACCTGTCTAGGTTTGATGC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-TGGCTTCATAGGTGACTTCCA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSCD1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-TCTAGCTCCTATACCACCACCA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-TCGTCTCCAACTTATCTCCTCC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLDLR\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-ACCAACGAATGCTTGGACAAC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-ACAGGCACTCGTAGCCGAT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eACACA\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-ATGTCTGGCTTGCACCTAGTA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CCCCAAAGCGAGTAACAAATTCT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGRP78\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CATCACGCCGTCCTATGTCG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CGTCAAAGACCGTGTTCTCG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eATF4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CGAGGTGTTGGTGGGGGACTTGA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CAACCCATCCACAGCCAGCCATT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eXBP1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-AACCTGTAGAAGATGACCTCGTTCC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-AAAGAGTTCATTGGCAAAAGTTCCAG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCHOP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CCCTCACTCTCCAGATTCCAGTC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CTAGCTGTGCCACTTTCCTTTCA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGAPDH\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-TCAAGAAGGTGGTGAAGCAGG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-TCAAAGGTGGAGGAGTGGGT-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eATF6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CTGATGGCTGTTCAATACACAG-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-GATCCCTTCGAAATGACACAAC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e33\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePERK\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CCAGTTTTGTACTCCAATTGCA-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5\\u0026rsquo;-CAGATACAGCTGGCCTCTATAC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e33\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMetabolic profiling\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eThe metabolic profiles of all samples were analyzed using the gas chromatograph/mass spectrometric (GC/MS) method, as described in previous studies [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. The measurements were carried out by BioNovoGene Company (Jiangsu, China). Chromatographic and spectral analysis was performed using ChemStation and MassHunter (Agilent Technologies).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eFlow cytometry analysis\\u003c/h3\\u003e\\n\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eCell surface markers were determined by staining with fluorochrome-conjugated monoclonal antibodies (mAbs). The antibody panel included PE-conjugated CD36 for the analysis of lipoprotein receptors. Briefly, cells were incubated with inhibitors and CBD as indicated above. Subsequently, the cells (1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells/tube) were resuspended in 50 \\u0026micro;l of PBS containing the recommended concentrations of antibodies according to the manufacturer's instructions, and then incubated for 30 minutes at 4\\u0026deg;C in the dark. Flow cytometry was conducted using a BD LSRFortessa, and the data was analyzed with FlowJo software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eEvaluation of cell cycle distribution and cell apoptosis by flow cytometry\\u003c/h3\\u003e\\n\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eFollowing treatment with CBD and the indicated inhibitors, PI staining was utilized for the analysis of DNA content. ES-2 cells were harvested and fixed with 70% ethanol at 4\\u0026deg;C overnight. Subsequently, Cell cycle and apoptosis kit was applied to assess DNA content using flow cytometry (BD Biosciences LSRFortessa, USA). The distribution of cells in the G0, G1, S, G2, and M phases was analyzed using Modfit 5.0 software.\\u003c/p\\u003e \\u003cp\\u003eThe percentage of cells undergoing apoptosis was determined by double staining with the FITC annexin V apoptosis detection kit and PI. Treated ES-2 cells were collected and resuspended in annexin V binding buffer. A 100 \\u0026micro;l cell suspension was transferred into a 1.5 ml tube, to which 5 \\u0026micro;l of FITC annexin V and 10 \\u0026micro;l of PI solution were added. After incubation for 15 minutes at 25\\u0026deg;C in the dark, 300 \\u0026micro;l of annexin V binding buffer was added to each tube. Flow cytometry was performed using a BD Biosciences LSRFortessa, and data were analyzed with FlowJo software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eJC-1 mitochondrial membrane potential (MMP) assay\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eThe MMP was evaluated using a mitochondria staining kit (Boyotime, Shanghai, China). The cells were seeded on 12-well plates and incubated with 5 mM NAC in 96-well plates at 37\\u0026deg;C and 5% CO2 for 30 min, followed by treatment with CBD at a final concentration of (30 \\u0026micro;M, 50 \\u0026micro;M) for 24 h. Subsequently, the cells were collected by centrifugation and resuspended in a staining solution containing 200 \\u0026times; JC-1 and 1 \\u0026times; staining buffer, then incubated at 37\\u0026deg;C for 20 min. Finally, the cells were washed once with JC-1 buffer. Data analysis was performed using Flowjo software. The MMP depolarization was visualized using a Leika fluorescence microscope (Leika, Wetzlar, Germany).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDetermination of cellular ROS\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eThe intracellular levels of reactive oxygen species (ROS) were analyzed using a 2\\u0026prime;,7\\u0026prime;-dichlorofluorescin diacetate (H2DCFDA) cellular ROS detection assay kit (KeyGen Biotech Co., Nanjing, China). After treatment with NAC and CBD as indicated above, supernatants were removed from the treated cells and replaced with 5 \\u0026micro;M DCFH-DA solution for 30 min at 37\\u0026deg;C in the dark. After incubation, the cells were washed three times with PBS to remove residual particles, dead cells, and excess DCFH-DA probes. The fluorescence intensity of ROS was measured at Ex/Em 488/525 nm by a microplate reader.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blotting\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eAfter treatment with CBD and inhibitors as described above, a total of 4 \\u0026times; 106 ES-2 cells were collected. The cells were then lysed in RIPA lysis buffer at 4\\u0026deg;C for 10 minutes to extract cellular protein. Samples containing equal amounts of protein (20\\u0026ndash;30 \\u0026micro;g) were mixed with 5 \\u0026times; Laemmli buffer, boiled, and separated on 10\\u0026ndash;15% SDS-PAGE gels. Samples were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk, the primary antibody was incubated overnight at 4\\u0026deg;C in an appropriate dilution. After washing, the blots were further incubated with HRP-conjugated secondary antibody for 1 h. Detection was performed using an enhanced chemiluminescence method. In our experiments, we used the following algorithm to evaluate the relative expression level of the target protein: Control group = [Control (Target protein/ Housekeeping protein) / Control (Target protein/Housekeeping protein)]\\u0026thinsp;=\\u0026thinsp;1.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll values are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD). The data were analyzed using a two-way analysis of variance (ANOVA) method. The post-hoc comparison was conducted using the follow-up least significant difference (LSD) test to assess differences between groups. Statistical significance was considered at a \\u003cem\\u003ep\\u003c/em\\u003e value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 level.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD inhibits OC cell proliferation via a CBR1-mediated mechanism\\u003c/h2\\u003e \\u003cp\\u003eFirst, we conducted the CCK-8 assay to investigate the impact of CBD on the viability of various types of OC cells (ES-2, SKOV3, Hey-A8). As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, CBD demonstrated significant antitumor activity in OC cells (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Moreover, at the same concentration, the antitumor efficacy of CBD was notably higher in ES-2 cells compared to SKOV3 and Hey-A8 cells (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The biological activity of CBD is known to be regulated by several receptors, including CBR1, CBR2, the transient receptor potential vanilloid subfamily 1 (TRPV1) receptor, and peroxisome proliferator-activated receptor γ (PPARγ) [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Next, we examined the relevance of these receptors to CBD-induced cell death by utilizing their selective inhibitors: AM251 (CB1 receptor antagonist), AM631 (CB2 receptor antagonist) [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e], and Capsazepine (TRPV1 receptor antagonist) [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e], and GW9662 (PPARγ inhibitor) [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. We observed that the CBD-induced inhibition of cell viability was reversed by pre-treatment with AM251, but not by AM631. In addition, treatment with Capsazepine or GW9662 alone did not have any effect on the viability of the tested OC cells (data not shown). Additionally, we also examined the expression of CBR1 in OC cells. In the interim, we also conducted an investigation on the expression of CBR1 in OC cells. The protein level of CBR1 was found to be significantly higher in ES-2 cells compared to SKOV3 and Hey-A8 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB and C). Additionally, we subjected ES-2 cells, which exhibited high sensitivity to CBD, to treatment with varying concentrations of CBD for 24 and 48 hours. The findings revealed that CBD (20\\u0026ndash;60 \\u0026micro;M) significantly hindered the proliferation of ES-2 cells in a manner dependent on both time and dosage. Furthermore, an IC\\u003csub\\u003e50\\u003c/sub\\u003e value of 32 \\u0026micro;M was observed after 24 hours of treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). These findings indicate that CBD may restrict the growth of OC cells in a manner dependent on CBR1.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD induced ER stress in OC cells\\u003c/h2\\u003e \\u003cp\\u003eThe DEGs were identified using RNA-seq.\\u0026nbsp;As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, a total of 2216 genes were found to be significantly upregulated, while 1908 genes were observed to be significantly downregulated following treatment with 40 \\u0026micro;M CBD in ES-2 cells. Upon Go analysis, it was revealed that the upregulated differentially expressed genes (DEGs) were highly relevant to the ER-associated misfolded protein catabolic process and PERK-mediated unfolded protein response pathways (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). We also observed that CBD-induced reduction in cell viability was reversed upon pre-treatment with 4-Phenylbutyric acid (4-PBA), a specific and irreversible inhibitor of ER stress, in a dose-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Subsequently, the transcriptome data was further validated using qRT-PCR, and the mRNA transcription levels of the genes related to ER stress were determined. The results demonstrated a significant increase in the mRNA transcription levels of glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), ATF6, PERK, ATF4, and CHOP in the treatment group compared to the control group (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The qRT-PCR results for the CBD-induced ER stress-related genes were in accordance with the findings from RNA-Seq analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). To investigate the intrinsic mechanism of this effect, western blot analysis was conducted. The findings indicated that CBD could significantly increase the mRNA transcription and protein expression of ER stress-related markers in a dose-dependent manner (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). 4-PBA effectively inhibited the upregulation of ER stress-related markers induced by CBD in a dose-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE-I). These findings suggest that CBD may suppress the growth of OC cells through the ER stress pathway.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD induces OC cells apoptosis and cell cycle arrest by regulating the ER stress signaling\\u003c/h2\\u003e \\u003cp\\u003eTo further investigate the correlation between apoptosis and CBD-induced ER stress in OC cells, Annexin V/PI staining was assessed using flow cytometry assay. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and B illustrate that CBD can effectively induce apoptosis in ES-2 cells in a manner that is dependent on dosage (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Furthermore, the addition of the ER stress inhibitor, 4-PBA groups, significantly prevented CBD-induced apoptosis. The observed increase in apoptosis due to CBD was consistent with an enhanced inhibition of tumor cell growth.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe utilized flow cytometry to further assess the impact of CBD on the distribution of cell cycles in OC cells. The exposure of these cells to CBD led to a dose-dependent increase in the percentage of cells in the G0-G1 phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC and D). Furthermore, the additional 4-PBA groups significantly rescued the CBD-induced G0-G1 phase cycle arrest of OC cells (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Based on these above-mentioned results, we have confirmed that CBD is capable of inducing apoptosis in OC cells and arresting the G0-G1 phase cycle through the activation of ER stress signaling.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD causes lipid metabolism disorder and induces ER stress response\\u003c/h2\\u003e \\u003cp\\u003eFatty acids serve as an important carbon source in the tumor microenvironment, and fatty acid-mediated lipid metabolism plays crucial roles in the survival of ovarian cancer cells [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. Elevated levels of unsaturated fatty acids (UFAs) have been shown to provide protection for cancer cells against apoptosis induced by ER stress [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. To investigate the impact of fatty acid metabolism on CBD-induced apoptosis in OC cells, we initially conducted a GC-MS analysis to assess the levels of lipid metabolites in CBD-treated OC cells. As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, the group treated with 40 \\u0026micro;M CBD showed significantly higher levels of saturated fatty acids (SFAs), including myristic acid (14C:0), palmitic acid (16C:0), and stearic acid (18C:0) compared to the control group (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Simultaneously, the levels of UFAs' content, particularly palmitoleic acid (C16:1), heptadecenoic acid (C17:1), and oleic acid (C18:1), were found to be significantly reduced following treatment with CBD in ES-2 cells (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Furthermore, the analysis using Go revealed that the downregulated DEGs are highly associated with the UFA metabolic process and fatty acid biosynthetic process (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). The results of qPCR analysis demonstrated that the CBD-treated group was able to reduce the transcription levels of genes associated with fatty acid uptake, including fatty acid binding protein 5 (FABP5), low-density lipoprotein receptor (LDLR), and PPARγ. The proportion of CD36-positive events was reduced following treatment with CBD (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC and D). Additionally, the transcription levels of genes related to fatty acid biosynthesis, such as acetyl-CoA carboxylase alpha (ACACA), Fatty acid synthase (FASN), SCD1, and sterol regulatory element binding protein 1 (SREBP), were also decreased post-CBD treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). Additionally, we investigated whether CBD could inhibit the uptake of fatty acids from the extracellular environment using fluorescently labeled palmitate (BODIPY FLC16) [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. The findings indicated that the CBD treatment group significantly inhibited the uptake of palmitate (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003ePrevious research has indicated that elevated levels of saturated fatty acids (SFAs), as a result of inhibiting stearoyl-CoA desaturase 1 (SCD1), can induce ER stress and apoptosis in several types of cancer. Furthermore, ER stress could be effectively alleviated through supplementation with UFAs [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. In this study, we observed that the addition of exogenous oleic acid could effectively alleviate the CBD-induced antitumor activities in OC cells in a dose-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF). CBD significantly inhibited the gene transcription and protein expression of SCD1 in a dose-dependent manner. Additionally, treatment with CBD resulted in a significant relief of both mRNA transcription and protein expression of ER stress-related markers by oleic acid in a dose-dependent manner (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG-N). Altogether, these data suggested that CBD interfered with lipid metabolism leading to ER stress in OC cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eOleic acid rescued CBD-directed ER stress-mediated apoptosis and cell cycle arrest\\u003c/h2\\u003e \\u003cp\\u003eTo determine whether UFAs provide broad protection against CBD-induced ER stress-related apoptosis, We utilized Annexin V/PI staining to assess the levels of apoptosis in ES-2 cells. The results are presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and B, CBD-treated group significantly increase apoptosis rates including both early and late apoptosis compared with the control group in serum-free medium. The CBD-induced apoptosis was significantly inhibited by supplying with OA (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Furthermore, exogenous OA had a marked effect on restoring the cell cycle porfile of CBD-treated cells in a dose-dependent manner (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC and D). From these results above, our findings have confirmed that the imbalance between unsaturated and saturated fatty acids is the primary cause of CBD-induced ER stress-related apoptosis in OC cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD induce lipid metabolism disorder and ER stress via a CBR1-mediated mechanism\\u003c/h2\\u003e \\u003cp\\u003ePrevious research has demonstrated that several crucial pathways involved in the growth, differentiation, and metabolism of tumor cells interact with CBR signaling [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. To examine whether CBD-induced fatty acid metabolism disorder and ER stress are CBR1-mediated, we used AM251, the antagonist of CBR1, to suppress the activity of CBR1 in ES-2 cells. As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, the CBD-induced inhibition of cell viability was found to be dose-dependently reversed by pre-treatment with AM251. Meanwhile, AM251 significantly rescued apoptosis and G0-G1 phase arrest of CBD-treated cells (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-D). In qPCR analyses, the treatment with CBD resulted in a significant dose-dependent decrease in the transcription levels of genes associated with fatty acid metabolism (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The downregulation of those genes were restored after blocking the CBR1 signaling with AM251 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe balance of SFAs and UFAs is regulated by SCD1 to finely tune the functions of ER stress [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Subsequently, we investigated the levels of gene transcription and protein expression associated with the ER response after blocking the CBR1. The upregulation of mRNA transcription and the protein expression of ER stress-related markers were significantly alleviated by AM251 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-I). These findings suggest that CBD possesses the capability to regulate disruption of lipid metabolism in OC cells and reduce UFA synthesis through the CBR1-SCD1 signaling pathway, ultimately resulting in ER stress-induced apoptosis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD induced mitochondrial dysfunction and cascade-mediated apoptotic pathways\\u003c/h2\\u003e \\u003cp\\u003eAlteration of ER stress markers is a key indicator of mitochondrial stress [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Based on the above findings, we hypothesized that CBD may induce cytotoxicity by targeting the mitochondria. CCK-8 and annexin V staining results indicated that the inhibitory effects of CBD on cell viability and apoptosis were significantly attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), a dose-dependent scavenger of reactive oxygen species (ROS) (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and B).\\u003c/p\\u003e \\u003cp\\u003ePrevious research has demonstrated that levels of MMP and ROS are commonly utilized as markers to assess mitochondrial function [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. To further investigate the effect of CBD on mitochondrial function, we first detected the MMP by the fluorescent probe JC-1. Comparative to the control group, treatment with CBD led to an increase in JC-1 monomer green fluorescence and a decrease in JC-1 aggregation (red fluorescence), resulting in a higher JC-1 monomer ratio. The flow cytometry results demonstrated that treatment of ES-2 cells with 30 and 50 \\u0026micro;M CBD led to a significant increase in the loss of MMP by 8.4 and 14.5-fold respectively, compared to DMSO-treated control cells. Furthermore, intracellular levels of ROS in CBD-treated ES-2 cells increased by up to 1.3 and 1.8-fold, respectively, in a dose-dependent manner compared to control cells. Similarly, the decrease in MMP and the generation of ROS could be significantly reversed by NAC treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA-C).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn addition, western blot revealed a significant downregulation of mitofusion 2 and antiapoptotic Bcl2 in the high CBD group, while the expression of cleaved Caspase-8, Caspase-9, and Caspase-3 was significantly upregulated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD-I). Taken together, our data showed that CBD may trigger mitochondrial dysfunction and apoptosis in OC cells.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eCBD is a non-addictive compound found in the medicinal plant Cannabis. Most previous studies have primarily focused on the protective effect of the nervous system, due to its ability to easily pass through the blood-brain barrier [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. Additionally, current evidence has demonstrated that CBD can inhibit the proliferation and metastasis of tumor cells, as well as induce apoptosis and autophagy in various types of cancer including breast, colorectal cancer, and gastric cancer [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. These studies also demonstrated that the involvement of ER stress or oxidative stress in the induction of cell death following treatment with CBD. CBD acts its effects mainly through the expression of CBD receptors on the cells.\\u003c/p\\u003e \\u003cp\\u003eSignificantly, high levels of CBR1 expression have been observed in ovarian cancer (OC) tissue, and the expression of CB1R is positively correlated with the level of malignancy in OCs [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Therefore, as a specific CBR1 agonist with efficacy against OC, CBD could be effectively utilized for OC therapy. In this study, we have observed high expression of CBR1 in all three types of human OC cells. However, a receptor-mediated mechanism contributing to the promising antitumor activity of CBD in OC cells has not yet been elucidated.\\u003c/p\\u003e \\u003cp\\u003eThe results of our study demonstrate a significant difference in the CBD-induced antitumor effect on OC cell lines ES-2, SKOV3, and Hey-A8. The inhibitory effects of CBD on ES-2 cells were found to be significantly higher (45.1%) compared to SKOV3 (25.46%) and Hey-A8 (21.29%). Additionally, the western blot results demonstrated a significantly higher protein level of CBR1 in ES-2 cells compared to that in SKOV3 and Hey-A8 cells. After inhibition of CBR1 by AM251, the CBD-induced repression of cell viability was significantly reversed. These results suggest that CBD may inhibit the growth of OC cells in a CBR1-mediated manner. To further elucidate the antitumor mechanism of CBD in OC cells, we initially utilized transcriptome analysis to examine the DEGs in ES-2 cells. Go analysis results suggest that CBD-induced antitumor effect was highly relevant to the ER-associated misfolded protein catabolic process. In addition, the gene transcript and expression levels for ER stress were significantly upregulated after treatment with CBD. Blocking ER stress response with 4-PBA, we revealed that CBD-induced OC cells apoptosis and G0-G1 phase cycle arrest relied on activation of XBP1 and ATF4/CHOP signaling pathways.\\u003c/p\\u003e \\u003cp\\u003eFatty acids play a crucial role as important energy sources and serve as a key component of phospholipids in cell membranes [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. In OCs, the accumulation of UFAs has been shown to support cell growth, as well as increase cancer cell migration and invasion [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. Lipid metabolism plays a complex role in multiple cell death pathways, including necroptosis and ferroptosis [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Recently, Zhao et al. revealed that controlling the balance between SFAs and UFAs can initiate ER stress in tumors [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. These findings suggest that regulating lipid metabolism in OCs may be a potential therapeutic strategy. To test this prediction, using GC-MS and RNA-seq analysis, we observed that the CBD group significantly downregulated the UFA metabolic process and decreased the transcription levels of genes associated with fatty acid uptake and synthesis in OC cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). CD36 regulates cellular energy homeostasis and intracellular cholesterol in OC cells [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Likewise, CD36 expression was significantly decreased after treatment with CBD. In addition, we found that CBD could convert SFAs to UFAs and palmitic and stearic acid to palmitoleic and OA, respectively. The regulation of the conversion from saturated fatty acids to unsaturated fatty acids is mediated by SCD1 [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Meanwhile, SCD1 and its predominant product, oleic acid, have been shown to have beneficial effects in restoring ER homeostasis, promoting cell cycle progression, and enhancing proliferation [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. This conclusion is further supported by our results, exogenous oleic acid could significantly relieve CBD-induced antitumor activities, apoptosis and G0-G1 phase arrest. Based on the combined results of qPCR and western blot analysis, it was observed that CBD-induced mRNA transcription and protein expression of ER stress-related markers could be significantly mitigated in a dose-dependent manner by exogenous oleic acid. These results strongly imply that CBD may initiate ER stress-associated apoptosis by disrupting lipid metabolism.\\u003c/p\\u003e \\u003cp\\u003eOver the past decade, numerous studies have demonstrated that CB1 and CB2 receptor agonists can function as direct antitumor agents by activating ERK, p38 MAPK, and JNK1/2 pathways [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. However, a clear distinction was observed between the activity produced and the specific cancer cell line studied. Furthermore, numerous studies have suggested that CBD may hinder the viability of cancer cells through mechanisms that bypass the activation of cannabinoid receptors. Ramer et al. reported that CBD induced PPARγ-dependent toxicity in lung cancer cells [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. Another study found CBD mediated endothelial cell autophagy and apoptosis via ROS-mediated heme oxygenase-1, but not by the CBD-activated receptors (CBR1, CBR2) [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. A recent investigation found that TRPV2 is a target of cannabinoids and is involved in CBD-induced autophagic death of glioma stem-like cells [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. In breast cancer cell lines, CBD induced ER stress through the TRPV1 receptor-dependent signaling pathway by increasing Ca\\u003csup\\u003e2+\\u003c/sup\\u003e influx [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. These results all show that CBD can modulate certain pathways involved in cancer development and exert their antitumor effects. Our study revealed that CBD exhibited very effective antitumor activity in OC cells and significantly downregulates the transcription of genes related to fatty acid metabolism via CBR1 signaling. Notably, we also observed that CBD significantly decreased the protein expression of p65, which is necessary for regulating SCD1 activity [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. The downregulation of CBD-induced p65 and SCD1were also relieved by blocking CBR1. Similarly, the upregulation of mRNA transcription and protein expression of ER stress-related markers was significantly alleviated by AM251. In accordance with our findings, a previous study has reported that increased expression levels of SCD1 protected ovarian cancer cells from apoptosis induced by ER stress [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Collectively, these results suggest that CBD may regulate lipid metabolism via CBR1/SCD1 signaling pathway to modulate ER stress-triggered apoptosis in OC cells.\\u003c/p\\u003e \\u003cp\\u003eMitochondria are essential for generating energy and play a critical role in maintaining cell survival and promoting metastatic evasion [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Mitochondrial dysfunction, which results in depolarized MMP and decreased ATP levels, suppresses OC progression [\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e]. Previous studies showed that mitochondria and ER crosstalk was also important in regulating cell metabolism and cell death [\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e]. The process of CHOP-mediated apoptosis involves the regulation of Bcl2 family proteins, leading to the induction of mitochondrial outer membrane permeabilization and caspase-dependent cell death [\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e]. In our study, flow cytometry analysis demonstrated that CBD could dose-dependently increase the loss of mitochondrial membrane potential (MMP). The decrease in MMP will consequently result in an elevation of the intracellular ROS level. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eC, the level of ROS content in the CBD-treated group was found to be higher than that in the control group. Once the MMP is fully depolarized, it will trigger caspase-dependent apoptosis pathways [\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e]. This process occurs within the mitochondria and plays a crucial role in programmed cell death. The conclusions were consistent with our western blot results. In this study, the expression levels of Bcl2 and mitofusion 2 were significantly downregulated and the pro-apoptotic protein caspase-8, -9, and \\u0026minus;\\u0026thinsp;3 were significantly upregulated after treatment with CBD in ES-2 cells. Meanwhile, CBD-induced mitochondria dysfunction and the expression of apoptotic proteins could be significantly reversed by NAC. These results indicate that CBD may trigger OC cells apoptosis through the ER-mitochondrial pathway.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eIn summary, our results demonstrated that CBD promoted OC cells apoptosis and G0-G1 phase arrest by disrupting the CBR1-mediated lipid metabolism and ER stress- and mitochondrial dysfunction-associated apoptosis signaling pathways (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). These findings could potentially contribute to the development of CBD as a pharmaceutical for treating OC.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eConflicts of interest\\u003c/h2\\u003e \\u003cp\\u003eThe authors declare no financial or commercial conflict of interest.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eConceptualization, X.F., and G.L.; Formal analysis, X.F., Z.Y., Y.B., W.Z., B.Y., Y.S., Z.J., and X.T.; Funding acquisition, G.L.; Investigation, X.F., Z.Y., X.T., Z.J., and B.Y.; Methodology, X.F., G.L. and Z.Y.; Project administration, G.L.; Software, X.F., F.F., Z.Y., B.Y. Y.S. and X.T.; Supervision, G.L.; Validation, G.L.; Writing \\u0026ndash; original draft, X.F.; Writing \\u0026ndash; review \\u0026amp; editing, G.L.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eThis research was supported by grant from the Natural Science Foundation of Liaoning Province of China (2022-MS-409), Science and Technology Innovation Project of Shenyang (RC210215).\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe transcriptome sequences of ES-2 (control) and treatment with CBD groups were deposited in NCBI under the accession number PRJNA980416.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBae, H., Song, G. \\u0026amp; Lim, W. Stigmasterol causes ovarian cancer cell apoptosis by inducing endoplasmic reticulum and mitochondrial dysfunction. Pharmaceutics. \\u003cstrong\\u003e12\\u003c/strong\\u003e,488. 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(2012).\\u003c/li\\u003e\\n\\u003cli\\u003eGharbaran, R., Shi, C., Onwumere, O. \\u0026amp; Redenti, S. Plumbagin induces cytotoxicity via loss of mitochondrial membrane potential and caspase activation in metastatic retinoblastoma. \\u003cem\\u003eAnticancer Res\\u003c/em\\u003e. \\u003cstrong\\u003e41\\u003c/strong\\u003e,4725-4732. (2021).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Ovarian cancer, Lipid metabolism, Cannabidiol\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5359456/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5359456/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eOvarian cancer (OC) is the most deadly gynecological tumor. OC cells utilize cellular metabolic reprogramming to gain a survival advantage, particularly through aberrant lipid metabolic process. As the primary ingredient in exogenous cannabinoids, cannabidiol (CBD) has been shown to exert anticancer effects in several cancers. However, it is still unclear whether CBD can disrupt fatty acid metabolism and induce apoptosis in OC cells. In this study, we have demonstrated that CBD significantly inhibits the proliferation of OCs through a dependence on cannabinoid receptor type 1 (CB1R). Lipidomics and flow cytometry analysis revealed that CBD has the ability to decrease fatty acid levels and significantly suppress the transcription of genes involved in fatty acid uptake and synthesis in ES-2 cells. In addition, the analysis from RNA-seq and real-time RT-PCR revealed that CBD activated the endoplasmic reticulum (ER) stress pathway. Conversely, by supplementation with unsaturated fatty acid or blocking CB1R, ER stress or reactive oxygen species (ROS) signals with specific inhibitors could significantly relieve CBD induced a dose-dependent ER stress associated apoptosis, G0-G1 phase arrest, and mitochondrial dysfunction. Taken collectively, these data indicate that CBD may disrupt lipid metabolism, and lead to ER stress-related apoptosis in OCs. Our findings may provide a theoretical mechanism for anti-ovarian cancer using CBD.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Cannabidiol attenuates lipid metabolism and induces CB1 receptor-mediated ER stress associated apoptosis in ovarian cancer cells\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-12-06 11:05:51\",\"doi\":\"10.21203/rs.3.rs-5359456/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-12-18T17:01:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-12-17T17:10:01+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-11-28T16:10:44+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-11-28T05:30:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"38229527601719362912983665971616191471\",\"date\":\"2024-11-27T19:38:48+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"76213844532615567605118561832319740746\",\"date\":\"2024-11-27T09:22:39+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"112646603896451574459448097898924761287\",\"date\":\"2024-11-16T11:45:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-11-15T17:33:14+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-11-15T17:24:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-11-13T06:38:33+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-11-13T06:36:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2024-10-30T07:52:20+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"3882a3df-606d-4fe6-9907-3faa5245bd3c\",\"owner\":[],\"postedDate\":\"December 6th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":40904069,\"name\":\"Biological sciences/Biochemistry\"},{\"id\":40904070,\"name\":\"Biological sciences/Cancer\"},{\"id\":40904071,\"name\":\"Biological sciences/Drug discovery\"},{\"id\":40904072,\"name\":\"Biological sciences/Immunology\"},{\"id\":40904073,\"name\":\"Biological sciences/Molecular biology\"},{\"id\":40904074,\"name\":\"Health sciences/Molecular medicine\"}],\"tags\":[],\"updatedAt\":\"2025-02-10T15:59:14+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5359456\",\"link\":\"https://doi.org/10.1038/s41598-025-88917-1\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2025-02-05 15:56:59\",\"publishedOnDateReadable\":\"February 5th, 2025\"},\"versionCreatedAt\":\"2024-12-06 11:05:51\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-025-88917-1\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-025-88917-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5359456\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5359456\",\"identity\":\"rs-5359456\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}